Laboratory Notes for BIO 1003

PHOTOSYNTHESIS

INTRODUCTION

Heterotrophic organisms such as fungi and bacteria obtain the energy they need for growth, reproduction, and movement by decomposing other organisms or molecules. Members of the Kingdom Plantae, together with some members of the Kingdom Protista and all of the cyanobacteria (Domain Bacteria), are photosynthetic organisms; as such, they are autotrophs: they synthesize their own food by using simple raw materials plus the energy of sunlight. They are responsible for renewing the energy supplies available on the Earth. Coal, gasoline, and heating oil fuel our society. These sources of energy are largely derived from the metabolic activity of plants that lived millions of years ago. Members of the Kingdom Animalia, heterotrophic organisms including ourselves, obtain energy from the food they eat. In addition, the process of photosynthesis is the source of oxygen required for the respiration of both plants and animals.

The process of photosynthesis transduces (converts) the kinetic energy of sunlight into the potential energy of chemical bonds. The energy is initially trapped in ATP molecules, later incorporated into the bonds of glucose, and eventually stored as carbohydrates—sugar or starch. Because this laboaratory exercise is about the Kingdom Plantae, the process of photosynthesis will be examined as it is carried out in the chloroplasts of plant cells.

The process of photosynthesis is a complex series of chemical reactions that begins with carbon dioxide and water (molecules of low potential energy) and ends with carbohydrates such as glucose and starch (molecules of high potential energy). The metabolic activity of plants enables the radiant energy of sunlight to be transduced (converted) to the energy found in the chemical bonds of carbohydrates.

Chlorophyll, the photosynthetic pigment in chloroplasts, absorbs light energy. Plants appear green because chlorophyll does not absorb light in the yellow-green region of the visible spectrum. Yellow and green wavelengths of the spectrum are reflected or transmitted depending on the thickness of a leaf. Chlorophyll absorbs light in the blue and red regions of the spectrum. The energy absorbed by chlorophyll raises the energy level of electrons in the active center of the chlorophyll molecule. In the chloroplast, the movement of these high-energy electrons is coordinated with the formation of the high-energy molecule, ATP, and the electron carrier, NADPH. During this process, water molecules are split and oxygen gas is released. This is origin of the oxygen in the air we breathe.

The importance of photosynthesis may be appreciated by considering the overall reaction:

ENZYMES

6CO2

+

6H2O

+

light

C6H12O6

+

6O2

carbondioxide

water

energy

glucose(food we eat)

oxygen(gas in the airwe breathe)

The overall process of photosynthesis has two parts, each consisting of groups of reactions with different purposes. The fluid inside a chloroplast is called stroma; the membranes inside the chloroplast in which the chlorophyll molecules are embedded are called thylakoids; in many places the thylakoids are closely stacked and appear as dark green dots or grana (grains) in each chloroplast.

1. The Light-Dependent Reactions require the presence of light. In green plants, these reactions take place within the chloroplast, and the enzymes that catalyze these reactions are integral parts of the thylakoid membranes, the membranes found within the chloroplast. As a result of the Light-Dependent Reactions, light energy is transduced to the chemical energy in the bonds of ATP. NADP+ is reduced when it picks up a pair of electrons to become NADPH. Water molecules are oxidized during the Light-Dependent Reactions and oxygen gas is released.

ENZYMES

ADP

+

phosphate group

+

light energy

ATP

ENZYMES

NADP+

+

electrons

+

hydrogen ion

+

light energy

NADPH

2. ATP and NADPH enter the Light-Independent Reactions. These reactions take place in the stroma of the chloroplast. This is the part of the interior of the chloroplast that is not occupied by the thylakoid membranes. During the Light-Independent Reactions, the energy associated with ATP and the hydrogen and electrons carried by NADPH are incorporated into a carbohydrate. This process is also called carbon dioxide fixation, meaning that carbon dioxide from the air is bound (fixed) into a stable carbohydrate. The simple carbohydrate that is formed may then be converted to glucose or to starch for storage. These reactions can be outlined as:

ENZYMES

a.

CO2

+

NADPH

+

ATP

G-3P

carbondioxide

electroncarrier

energysource

glyceraldehyde-3 phosphate(simple carbohydrate)

ENZYMES

ENZYMES

b.

simple carbohydrate

glucose

starch

LABORATORY EXERCISES

The goals of the laboratory exercises are:

to examine the structure of the leaf, a major site of photosynthesis in terrestrial plants;

to extract the photosynthetic pigment from the leaf and study some of its properties;

to test for photosynthetic products in a leaf.

1. LEAF STRUCTURE

The various arrangements of leaves on the stem ensure a minimum of shading of one another and a maximum of light absorption. The structure of an individual leaf is related to the functions it carries out. There is a large surface area that allows maximum absorption of light and an open spongy network of cells that allows gaseous interchange of water, carbon dioxide, and oxygen. However, this arrangement poses a problem for the plant because the large surface area allows water to be lost by evaporation. What helps retard water loss from the leaf?

A. Epidermal Structure of the Lilac Leaf (Syringa)

Obtain a slide of leaf epidermis. The lower epidermis has been stripped from a leaf, mounted on a slide, and then stained. Note the large clear epidermal cells. They have not been sectioned. By focusing up and down, the three-dimensional structure of the cells and how they interlock can be seen. The guard cells look rather banana-shaped in surface view. Because of their chloroplasts, they stain more darkly than the epidermal cells. The guard cells surround the stomates, small openings through which gas exchange occurs. Four modified epidermal cells, called supporting cells, surround the guard cells. Draw a diagram of the epidermis and label stomates, guard cells, and epidermal cells.

B. Cross Section of the Lilac leaf (Syringa)

Obtain a prepared slide of the leaf cross section. First hold it against a sheet of white paper. Note that there are several slices on the slide, each of which is quite narrow (from top to bottom). Compare the thickness of the slice to the thickness of a living leaf, for example, on the Coleus plant. Then examine the slide microscopically first under low and then high power. The leaf has an upper epidermis and a lower epidermis, both covered by an outer cuticle. The cuticle is composed of a waxy substance that helps cut down on water loss from the surface of the leaf.

Scan the lower epidermis and find the stomates surrounded by the guard cells. Note that the guard cells have chloroplasts. What is the function of guard cells? How do they operate? How might the presence of chloroplasts relate to the ability of the guard cells to open and close the stomate?

Explore the upper epidermis; are there any stomates here? (Do not confuse an occasional pit gland with a stomate.) The stomate leads to a substomatal cavity that is continuous with the air spaces in the middle region of the leaf. The middle of the leaf contains photosynthetic tissue (mesophyll) interspersed with the vascular tissue of the veins. Look at the arrangement of the photosynthetic cells of the middle region of the leaf. Notice that the cells of the upper layer (the palisade mesophyll) are very close to each other while there are much more extensive air spaces among the cells of the lower layer (the spongy mesophyll).

Locate the section of a large main vein. (For reference, look at a Coleus leaf and identify a main vein macroscopically.) Identify the thick-walled, large xylem vessels. Xylem vessels are the remains of cells that have lost their nuclei and cytoplasm but still have their thick cell walls. The vessels are arranged end-to-end like pipes. The phloem cells are smaller, thinner-walled cells. With the upper epidermis of the leaf at the top of the section, the position of the xylem in the vascular bundle is above the phloem. Xylem transports water and minerals from the root throughout the plant while phloem transports manufactured organic substances away from the leaf. The vascular tissue is surrounded by a sheath of large soft-walled cells (parenchyma cells). Thick-walled supporting cells (sclerenchyma) are found above and below the vascular bundle. The sclerenchyma extends all the way to the epidermis. Locate sections of smaller veins. Which tissue is missing?

In the space below, make a diagram of a cross section of the leaf and label cuticle, upper epidermis, chloroplasts in cells of the mesophyll, air spaces, xylem, phloem, guard cells surrounding a stomate, lower epidermis.

2. EXTRACTION AND SPECTRAL PROPERTIES OF CHLOROPHYLL

A. Before extracting chlorophyll from the cells of a Coleus leaf, note that the leaves contain other pigments in addition to chlorophyll. Observe the leaf and sketch its pigment pattern.

Boil some leaves in a small amount of water in a beaker on the hot plate. Does the water change color? What has happened?

Carefully pour off (decant) the hot water from the beaker and cover the leaf with alcohol. Boil the leaf gently (Why should the heat be set lower than for water?) Does the alcohol become green? What has happened? What does this indicate about the nature of chlorophyll? What has happened to the red pigment?

Carefully pour the colored alcohol solution into a test tube and save the leaf for Part 3.

Use the spectroscope to examine the spectrum of the chlorophyll. Examine the complete spectrum of white light in the spectroscope and note that it is broadly divided into a red band, a yellow-green, and a blue band. Now place a tube containing a fairly concentrated solution of chlorophyll between the spectroscope slit and the lamp. (If it is concentrated enough, use the solution saved in PART B above.) Which light bands are strongly absorbed? Which are transmitted most? What does this indicate about the function of chlorophyll? Would plants grow indoors using only a green light source?

B. What happens to the radiant energy absorbed by the chlorophyll in the solution? Fluorescence occurs when molecules absorb light of one wavelength and then emit light of a longer wavelength; this property can be observed in chlorophyll.

Place a solution of chlorophyll in a beaker and shine a narrow beam of light through the beaker. Look along the path of the beam of light within the solution. What is the color of the solution along the path of the beam of light? (Darken the room if necessary to ensure that the path of the beam of light is visible in the solution.)

In the absence of the organized membranes of the chloroplast, light absorbed by chlorophyll molecules in solution is given up and emitted as light of a longer wavelength than that originally absorbed. Look into the beaker and notice that red light (long wavelengths) is visible along the path of the light beam. What color(s) can chlorophyll absorb?

The chloroplasts of the leaf have organized membranes (the thylakoids) which contain the molecules needed to convert light energy into chemical energy. When light shines on chloroplasts, ATP and NAPDH molecules will be formed.

3. STARCH FORMATION IN THE LEAF

Starch is an end product of photosynthesis produced during the light-independent reactions. Starch is easily detected: Lugol's Iodine Reagent, which is dark amber colored, turns blue black in the presence of starch (see Organic Molecules lab).

Take the leaves that were boiled in alcohol, gently wash away the alcohol, and carefully flatten out each leaf in a Petri dish.

Cover the leaf with Lugol's Iodine Reagent. After one minute gently wash away any excess iodine.

What color was the iodine solution before it was put on the leaf?

What color does the leaf become?A blue-black color indicates that starch has combined with the iodine in the test reagent.

Compare the pattern of staining with the original pigment pattern sketched at the start.

Does the leaf stain positively for starch all over? If not, what does this indicate about the photosynthetic activity in a Coleus leaf?

Based on these tests, is the red pigment, observed originally in the Coleus leaf, a photosynthetic pigment?

Is the green pigment a photosynthetic pigment?

Look at the leaf under the microscope. Are there stained starch grains?

Thanks to Profs. Lea Bleyman, Sy Schulman, and Emil Gernert for permission to adapt the Photosynthesis Exercise from BIO 1005 for use in BIO 1003.